The de novo biosynthesis of triacylglycerol has been shown to occur by the sequential acylation of glycerol-3-phosphate (1–3). Glycerol-3-phosphate acyltransferase catalyzes the first step in glycerolipid synthesis (4) generating lysophosphatidic acid (LPA). Alternatively, LPA is formed by acylation followed by reduction of dihydroxyacetone phosphate that are catalyzed by dihydroxyacetone phosphate acyltransferase (5) and acyl-dihydroxyacetone phosphate reductase (6, 7), respectively. The acylation of LPA is catalyzed by LPA acyltransferase to form phosphatidic acid (PA), which is a branch point for the synthesis of diacylglycerol (DAG) and phospholipids. PA phosphatase catalyzes the dephosphorylation of PA to DAG that is an immediate precursor for triacylglycerol (TAG), phosphatidylcholine and phosphatidylethanolamine. DAG can also be derived from phospholipids by the action of phospholipase C (8), which is an important signal molecule that activates protein kinase C (9). DAG acyltransferase catalyzes the acylation of DAG, which is the committed step in TAG biosynthesis. Recently, it has been shown in plants and yeast cells that an acyl-CoA independent enzyme for TAG synthesis that uses phospholipid as acyl donor and DAG as acyl acceptor. This reaction is catalyzed by phospholipid:DAG acyltransferase (10). The same reaction is also catalyzed by lecithin:cholesterol acyltransferase (11). All the enzymes in these pathways are shown to be membrane-bound in eukaryotic systems (1–4, 12, 13). Both mitochondrial membranes and endoplasmic reticulum (ER) have been identified as the major sites for phospholipid and TAG synthesis in S. cerevisiae (3, 6, 14).
A number of fungi are known to have high levels of TAG. Understanding the lipid biosynthesis would enable to genetically engineer fungi and plants with desired fatty acid composition and the altered oil content (15).
While there is no direct evidence to support the possibility that membranes are the only sites for TAG synthesis, there is also no evidence for the absence of this biosynthetic pathway in the cytosol. The presence of soluble enzymes that provide important precursors for triacylglycerol biosynthesis is well documented.
TAG enzymes in yeast comprise of lysophosphatidic acid acyltransferase, phosphatidic acid phosphatase, diacylglycerol acyltransferase, acyl—acyl carrier protein synthetase and acyl carrier protein. These TAG biosynthetic enzymes may exist as either free or multienzyme complex. Among these enzymes, lysophosphatidic acid acyltransferase can be identified in any yeast strain by immunological cross reactivity to the peptide sequence ALELQADDFNK (peptide SEQ ID NO: 2), diacylglycerol acyltransferase identified by immunological cross reactivity to the peptide sequence XLWAVVGAQPFGGARGS (peptide SEQ ID NO: 7) and phosphatidic acid phosphatase identified by immunological cross reactivity to peptides NALTGLHMGGGK (peptide SEQ ID NO: 4) and YVEGARP (peptide SEQ ID NO: 6). TAG biosynthetic enzymes thus isolated from oleaginous yeast can utilize free fatty acids or fatty acyl-CoA or acyl-ACP as substrates.
In oleaginous yeasts and other lipid rich fungi, biochemical and genetic study of these enzyme systems would enable to genetically engineer fungi and plants with desired fatty acid composition and altered oil content. Using modern methods of genetics, these enzymes may be produced by recombinant gene expression; the recombinant proteins may be reconstituted to active TAG biosynthetic complex; the complex may be suitably assayed to identify specific inhibitors of TAG biosynthesis, which may have a potential value as lipid lowering drugs in humans.
Keeping with and to further studies conducted, Applicant investigated cytosolic TAG biosynthetic pathway using Rhodotorula glutinis and other Saccharomyces cerevisiae, rat adipocytes and human hepatocytes cell-line.